Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems.

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

Closing the gap: An urgent need for newborn screening of organic acid disorders in developing countries.

Organic acid disorders are rare inherited metabolic disorders of key metabolic pathways. For the identification of specific organic acids, investigation of urinary metabolites and genetic testing are required through newborn screening programmes. Delayed diagnosis leads to complications, such as cardiac attacks, respiratory problems, neuro-developmental disorders, intellectual disability, and even premature death. The burden of such inherited disorders is quite high in developing countries of South Asia due to high rate of consanguinity in the region. Unfortunately, such disorders are left untreated due to the lack of screening facilities in such countries. The current narrative review was planned to highlight the urgent need for closing this gap and implementing effective newborn screening programmes for organic acid disorders in developing countries. The implementation of effective programmes is crucial for reducing morbidity and mortality, and for improving the quality of life for the affected children and of their families, thus promoting global health equity.

Mass-spectrometric analysis of APOB polymorphism rs1042031 (G/T) and its influence on serum proteome of coronary artery disease patients: genetic-derived proteomics consequences.

Genetic polymorphisms of apolipoprotein B gene (APOB) may result into serum proteomic perturbance in Coronary Artery Disease (CAD). The current case-control cohort of Pakistani subjects was designed to analyze the genetic influence of APOB rs1042031, (G/T) genotype on serum proteome. Subjects were categorized into two groups: CAD patients (n = 480) and healthy individuals (n = 220). For genotyping, tetra ARMS-PCR was carried out and validated through sequencing, whereas LC/MS-based proteomic analysis of serum samples was performed through label-free quantification. In initial step of genotyping, the frequencies of each genotype GG, GT, and TT were 70%, 27%, and 30% in CAD patients, while in control group, the subjects were 52%, 43%, and 5%, respectively, in CAD patients. The genotypic frequencies in patients vs. control groups found significantly different (p = 0.004), and a strong association of dominant alleles GG with the CAD was observed in both dominant (OR: 2.4 (1.71-3.34), p = 0.001) and allelic genetic models (OR: 2.0 (1.45-2.86), p = 0.001). In second step of label-free quantitation, a total of 40 significant proteins were found with altered expression in CAD patients. The enriched Gene Ontology (GO) terms of molecular functions and pathways of these protein showed upregulated pathways as follows: chylomicron remodeling and assembly, complement cascade activation, plasma lipoprotein assembly, apolipoprotein-A receptor binding, and metabolism of fat-soluble vitamins in G allele carrier of rs1042031 (G > T) vs. mutant T-allele carriers. This study provides better understanding of CAD pathobiology by proteogenomics of APOB. It evidences the influence of APOB rs1042031-dominant (GG) genotype with CAD patients.

Highly selective enrichment of phosphopeptides using poly(dibenzo-18-crown-6) as a solid-phase extraction material.

A poly(dibenzo-18-crown-6) was used as a new solid-phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo-18-crown-6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α- casein, ß-casein and ovalbumin) was applied on poly(dibenzo-18-crown-6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Highly selective enrichment of both mono- and multiphosphorylated peptides was achieved using poly(dibenzo-18-crown-6) as solid-phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo-18-crown-6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo-18-crown-6) and various ligands. The newly identified solid-phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.

In vitro photoprotective potential of aryl-sandwiched (thio)semicarbazones against UVA mediated cellular and DNA damage.

The most prevalent solar ultraviolet radiation is ultraviolet-A (UVA) radiation. It is the inducer of reactive oxygen species (ROS), a potent mediator of inflammation and photocarcinogenesis. Regular application of sunscreens containing UVA filters is an effective preventive measure in mitigating the risk associated with the formation of dermal carcinoma. Therefore, the development of new photoprotective agents is of great need. The current work examined the in vitro photoprotection of the aryl-linked (thio)semicarbazone derivatives against UVA-mediated DNA damage, inflammation, reactive nitrogen species (RNS), and ROS. Except for the inflammatory cytokine assay, which was carried out on the human monocytic leukemia (THP-1) cell line, all tests were conducted on the human dermal fibroblast (BJ) cell line. In comparison to benzophenone (reference compound), the compound (2Z, 2'Z)-2,2'-(1,3-Phenylenebis (methanylylidene)) bis (hydrazine-1-carbothioamide) (DD-21) demonstrated considerable protection against UVA-induced damage. Compared to the UVA-irradiated control, DD-21 significantly decreased the levels of nitric oxide (NO) and ROS (p < 0.001). In the presence of DD-21, the release of UVA-induced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), was also significantly reduced (p < 0.05). Moreover, it was observed that DD-21 protected the cells from UVA-mediated DNA strand breaks and also inhibited the formation of cyclobutane pyrimidine dimers (CPDs) upon comparison to the UVA-exposed control cells (p < 0.001). In conclusion, the findings of this study revealed that DD-21 exhibits remarkable photoprotective properties, thus demonstrating its potential as a candidate UVA filter.

Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-ß) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

Yellow scorpion (Buthus sinidicus) venom peptides induce mitochondrial-mediated apoptosis in cervical, prostate and brain tumor cell lines.

Scorpion venoms are known to contain over 100,000 biologically active constituents. However, only a few of them have been studied. The major constituents of venom are proteins and peptides, which exhibit various biological and pharmacological properties, including anticancer activities. In the current study, the venom of yellow scorpions (Buthus sindicus) found in Sindh, Pakistan, was extracted and evaluated for its anti-cancer and anti-inflammatory activities. The crude venom showed a dose dependent inhibition of phagocyte oxidative burst from human whole blood cells (28.3% inhibition at highest tested concentration of 300 µg/mL). In-vitro cytotoxicity of crude venom was evaluated against human prostrate (PC3), cervical (HeLa) and neuroblastoma (U87-MG) cell lines, along with cytotoxicity against normal human fibroblast (BJ) cells. Crude venom was cytotoxic to all cell lines, with prominent inhibitory effect on PC3 cells. Crude venom was fractionated through RP-UPLC, resulted in fifteen fractions, followed by evaluation of their anticancer potential. Among all, the fraction I significantly (P < 0.001) reduced the cell viability of all three cancer cell lines, and exhibited insignificant cytotoxicity against normal cell line. Furthermore, the apoptotic cell death pathway was evaluated for crude venom, and fraction I, in most sensitive cell line PC3, by using flow-cytometry analysis. Both crude venom and its fraction I caused a mitochondrial-mediated apoptosis in prostate cancer cells (PC3). To the best of our knowledge, this is the first report of the anticancer and anti-inflammatory activity of venom of Pakistani yellow scorpions. Results indicate their therapeutic potential, and a case for further purification and validation studies.

Highly efficient precipitation of phosphoproteins using trivalent europium, terbium, and erbium ions.

This study describes a highly efficient method for the selective precipitation of phosphoproteins by trivalent europium, terbium, and erbium metal ions. These metal cations belong to the group of lanthanides and are known to be hard acceptors with an overwhelming preference for oxygen-containing anions such as phosphates to which they form very tight ionic bonds. The method could be successfully applied to specifically precipitate phosphoproteins from complex samples including milk and egg white by forming solid metal-protein complexes. Owing to the low solubility product of the investigated lanthanide salts, the produced metal-protein complexes showed high stability. The protein pellets were extensively washed to remove nonphosphorylated proteins and contaminants. For the analysis of proteins the pellets were first dissolved in 30 % formic acid and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. For peptide mass-fingerprint analysis the precipitated phosphoproteins were enzymatically digested using microwave-assisted digestion. The method was found to be highly specific for the isolation and purification of phosphoproteins. Protein quantification was performed by colorimetric detection of total precipitated phosphoproteins and revealed more than 95 % protein recovery for each lanthanide salt.

A novel strategy for phosphopeptide enrichment using lanthanide phosphate co-precipitation.

Reversible phosphorylation of proteins is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation. The comprehensive understanding of biological processes requires the characterization of protein phosphorylation at the molecular level. Although, the number of cellular phosphoproteins is relatively high, the phosphorylated residues themselves are generally of low abundance due to the sub-stoichiometric nature. However, low abundance of phosphopeptides and low degree of phosphorylation typically necessitates isolation and concentration of phosphopeptides prior to mass spectrometric analysis. In this study, we used trivalent lanthanide ions (LaCl(3), CeCl(3), EuCl(3), TbCl(3), HoCl(3), ErCl(3), and TmCl(3)) for phosphopeptide enrichment and cleaning-up. Due to their low solubility product, lanthanide ions form stable complexes with the phosphate groups of phosphopeptides and precipitate out of solution. In a further step, non-phosphorylated compounds can easily be removed by simple centrifugation and washing before mass spectrometric analysis using Matrix-assisted laser desorption/ionisation-time of flight. The precipitation method was applied for the isolation of phosphopeptides from standard proteins such as ovalbumin, α-casein, and ß-casein. High enrichment of phosphopeptides could also be achieved for real samples such as fresh milk and egg white. The technology presented here represents an excellent and highly selective tool for phosphopeptide recovery; it is easily applicable and shows several advantages as compared with standard approaches such as TiO(2) or IMAC.

Probing photoprotection properties of lipophilic chain conjugated thiourea-aryl group molecules to attenuate ultraviolet-A induced cellular and DNA damages.

Ultraviolet-A (UVA) radiation is a major contributor to reactive oxygen species (ROS), reactive nitrite species (RNS), inflammation, and DNA damage, which causes photoaging and photocarcinogenesis. This study aimed to evaluate the UVA protective potential of lipophilic chain conjugated thiourea-substituted aryl group molecules against UVA-induced cellular damages in human dermal fibroblasts (BJ cell line). We tested a series of nineteen (19) molecules for UVA photoprotection, from which 2',5'-dichlorophenyl-substituted molecule DD-04 showed remarkable UVA protection properties compared to the reference (benzophenone). The results indicate that DD-04 significantly reduced intracellular ROS and nitric oxide (NO) as compared to the UVA-irradiated control (p < 0.001). Moreover, the compound DD-04 showed anti-inflammatory activity as it significantly reduced the levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) pro-inflammatory cytokines produced by THP-1 (human monocytic) cells (p < 0.05). DNA damage was also prevented by DD-04 treatment in the presence of UVA. It was observed that DD-04 significantly reduced the number of cyclobutane pyrimidine dimers (CPDs) when compared to the UVA-irradiated control (p < 0.001). Finally, the DNA strand breaks were checked and a single intact DNA band was seen upon treatment with DD-04 in the presence of UVA. In conclusion, DD-04 can be considered a potential candidate UVA filter due to its photoprotective potential.

Label-free quantitation of the changes in salivary proteome associated with the chronic consumption of the betel nut (Areca catechu).

Areca nut or betel nut chewing is most frequently used in Pakistan and is associated with a high risk for oral cancer. Until now, however, there has not been any research conducted on the long-term effect(s) of betel nut chewing on the saliva proteome. In the present study, initially, the changes in the saliva proteome associated with betel nut chewing were investigated. Secondly, the analysis was focused on the changes in salivary proteome with respect to prolonged usage of betel nuts. After extraction, the saliva proteins were digested into peptides and these were subsequently analyzed using mass spectrometry. Data are available via ProteomeXchange with identifier PXD029768. Label-free quantitation of saliva samples revealed a total of 12 proteins that were differentially expressed between betel nut addicts (BNAs), and the control group. The study groups were further divided into three subgroups, the BNA-1, BNA-2, and BNA-3 groups, with respect to the extent of consumption of betel nuts in terms of years. The data analysis revealed a more detailed profiling of proteins expressed after five, ten, and more than ten years of betel nut consumption. A total of 30, 17, and 22 proteins were found to be differentially expressed when divided into the BNA-1, BNA-2, and BNA-3 groups. The present study shows that the chronic usage of betel nuts leads to the expression of proteins, such as SPARC1, profilin, and SBSN, which are known to be involved in head and neck cancers.

Salmonella Typhi from Northwest Pakistan: Molecular Strain Typing and Drug Resistance Signature.

Objective: To determine the molecular strain typing and drug resistance pattern of Salmonella enterica serovar Typhi prevalent in Northwest Pakistan. Methodology: A total of 2,138 blood samples of suspected typhoid patients from Northwest Pakistan were collected followed by identification of Salmonella Typhi through biochemical, serological, and species-specific fliC-d gene amplification. These isolates were typed by variable-number tandem repeat (VNTR) profiling and investigated for drug resistance. Results: The overall prevalence of Salmonella Typhi was found to be 8.8% (n = 189). Thirty different VNTR strain types of Salmonella Typhi were detected and the most prevalent strain types were T1 and T4, whereas T27 was less prevalent strain. Among the 189 isolates 175 (92.5%) isolates were multidrug resistant, whereas 12 (5.8%) isolates were extensively drug resistant. Resistance to imipenem in Salmonella Typhi was not observed. Most of the isolates have genes encoding for resistance to fluoroquinolones, including gyrA (n = 164), gyrB (n = 160), parC (n = 164), parE (n = 160), ac(6')-ib-cr (n = 163), qnrS (n = 15), and qnrB (n = 3). Similarly, chloramphinicol (cat; n = 147), azithromycin (msrA; n = 3), and co-trimoxazole (dfrA7; n = 145) resistance genes were detected among Salmonella Typhi isolates. Conclusion: In this study, T1 and T4 type Salmonella Typhi strains were predominantly prevalent in Northwest Pakistan. Antibiotic resistance among Salmonella Typhi isolates were observed. Findings of the study would be helpful to devise an appropriate antibiotic policy to control the emergence of drug-resistant Salmonella Typhi in Pakistan.

Effect of APOB polymorphism rs562338 (G/A) on serum proteome of coronary artery disease patients: a "proteogenomic" approach.

In the current study, APOB (rs1052031) genotype-guided proteomic analysis was performed in a cohort of Pakistani population. A total of 700 study subjects, including Coronary Artery Disease (CAD) patients (n = 480) and healthy individuals (n = 220) as a control group were included in the study. Genotyping was carried out by using tetra primer-amplification refractory mutation system-based polymerase chain reaction (T-ARMS-PCR) whereas mass spectrometry (Orbitrap MS) was used for label free quantification of serum samples. Genotypic frequency of GG genotype was found to be 90.1%, while 6.4% was for GA genotype and 3.5% was for AA genotypes in CAD patients. In the control group, 87.2% healthy subjects were found to have GG genotype, 11.8% had GA genotype, and 0.9% were with AA genotypes. Significant (p = 0.007) difference was observed between genotypic frequencies in the patients and the control group. The rare allele AA was found to be strongly associated with the CAD [OR: 4 (1.9-16.7)], as compared to the control group in recessive genetic model (p = 0.04). Using label free proteomics, altered expression of 60 significant proteins was observed. Enrichment analysis of these protein showed higher number of up-regulated pathways, including phosphatidylcholine-sterol O-acyltransferase activator activity, cholesterol transfer activity, and sterol transfer activity in AA genotype of rs562338 (G>A) as compared to the wild type GG genotype. This study provides a deeper insight into CAD pathobiology with reference to proteogenomics, and proving this approach as a good platform for identifying the novel proteins and signaling pathways in relation to cardiovascular diseases.

Prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw foods in Peshawar, Pakistan.

This study was designed to investigate the prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw food samples in Peshawar, Pakistan. A total of 1,020 different samples were collected from various areas of Peshawar between January 2016 and May 2017, followed by identification of S. flexneri through biochemical, serological, and 16S rRNA gene sequencing. Potential risk factors associated with the development and spreading of S. flexneri infection were also investigated. Overall, 45 (4.41%) samples were positive for Shigella species. Among these samples, the predominant species was S. flexneri (n = 44) followed by S. boydii (n = 1). Interestingly, S. sonnei and S. dysenteriae isolates were not found in any sample. The isolation rate of S. flexneri in drinking water samples, market raw milk, and fruits/vegetables from Peshawar were 6.47%, 3.5%, and 2.9%, respectively. The phylogenetic reconstruction showed genetic diversity among three clades, as clades I and II have isolates of S. flexneri that were circulating within the drinking water, milk, fruits/vegetables, while clade III isolates were recovered from milk samples. Most of S. flexneri were detected in June to September. Potential risk factors of S. flexneri were water sources contaminated by toilet wastes (p = 0.04), surface water drainage (p = 0.0002), hospital wastes (p = 0.01), unhygienic handling (p < 0.05), and transportation of raw food (p = 0.04). In conclusion, S. flexneri isolates of closely related lineage originating from non-clinical samples might be associated with an increased human risk to shigellosis in Pakistan, as significant numbers of S. flexneri were observed in the drinking water and retail raw food samples. PRACTICAL APPLICATION: This study demonstrated the presence of S. flexneri in drinking water and retail raw food samples which seem to possess a serious threat to public health. Potential sources of food and water contamination should properly be monitored by public health authorities to reduce cases of shigellosis.

Bergenin protects pancreatic beta cells against cytokine-induced apoptosis in INS-1E cells.

Beta cell apoptosis induced by proinflammatory cytokines is one of the hallmarks of diabetes. Small molecules which can inhibit the cytokine-induced apoptosis could lead to new drug candidates that can be used in combination with existing therapeutic interventions against diabetes. The current study evaluated several effects of bergenin, an isocoumarin derivative, in beta cells in the presence of cytokines. These included (i) increase in beta cell viability (by measuring cellular ATP levels) (ii) suppression of beta cell apoptosis (by measuring caspase activity), (iii) improvement in beta cell function (by measuring glucose-stimulated insulin secretion), and (iv) improvement of beta cells mitochondrial physiological functions. The experiments were carried out using rat beta INS-1E cell line in the presence or absence of bergenin and a cocktail of proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon- gamma) for 48 hr. Bergenin significantly inhibited beta cell apoptosis, as inferred from the reduction in the caspase-3 activity (IC50 = 7.29 ± 2.45 µM), and concurrently increased cellular ATP Levels (EC50 = 1.97 ± 0.47 µM). Bergenin also significantly enhanced insulin secretion (EC50 = 6.73 ± 2.15 µM) in INS-1E cells, presumably because of the decreased nitric oxide production (IC50 = 6.82 ± 2.83 µM). Bergenin restored mitochondrial membrane potential (EC50 = 2.27 ± 0.83 µM), decreased ROS production (IC50 = 14.63 ± 3.18 µM), and improved mitochondrial dehydrogenase activity (EC50 = 1.39 ± 0.62 µM). This study shows for the first time that bergenin protected beta cells from cytokine-induced apoptosis and restored insulin secretory function by virtue of its anti-inflammatory, antioxidant and anti-apoptotic properties. To sum up, the above mentioned data highlight bergenin as a promising anti-apoptotic agent in the context of diabetes.